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An Arabidopsis gene regulatory network for secondary cell wall synthesis

Abstract

The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here we present a protein–DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

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Figure 1: Regulators of xylem development and secondary cell wall biosynthesis.
Figure 2: E2Fc represses secondary cell wall gene biosynthesis.
Figure 3: Tissue-specific VND7 regulation and VND7 targets.
Figure 4: Multiple transcription factors bind the CESA4 promoter.
Figure 5: The xylem-specific gene regulatory network is responsive to high salinity and iron deprivation.

References

  1. Brown, D. M., Zeef, L. A. H., Ellis, J., Goodacre, R. & Turner, S. R. Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell 17, 2281–2295 (2005)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Persson, S., Wei, H., Milne, J., Page, G. P. & Somerville, C. R. Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets. Proc. Natl Acad. Sci. USA 102, 8633–8638 (2005)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. Carlsbecker, A. et al. Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465, 316–321 (2010)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Mitsuda, N., Seki, M., Shinozaki, K. & Ohme-Takagi, M. The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17, 2993–3006 (2005)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Brady, S. M. et al. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318, 801–806 (2007)

    ADS  CAS  PubMed  Article  Google Scholar 

  6. Zhong, R., Lee, C., Zhou, J., McCarthy, R. L. & Ye, Z.-H. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20, 2763–2782 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Gaudinier, A. et al. Enhanced Y1H assays for Arabidopsis. Nature Methods 8, 1053–1055 (2011)

    CAS  PubMed  Article  Google Scholar 

  8. Kim, W.-C., Ko, J.-H. & Han, K.-H. Identification of a cis-acting regulatory motif recognized by MYB46, a master transcriptional regulator of secondary wall biosynthesis. Plant Mol. Biol. 78, 489–501 (2012)

    CAS  PubMed  Article  Google Scholar 

  9. Yamaguchi, M. et al. VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation. Plant J. 66, 579–590 (2011)

    CAS  PubMed  Article  Google Scholar 

  10. Zhou, J., Lee, C., Zhong, R. & Ye, Z. H. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21, 248–266 (2009)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Hussey, S. G., Mizrachi, E., Creux, N. M. & Myburg, A. A. Navigating the transcriptional roadmap regulating plant secondary cell wall deposition. Front. Plant Sci. 4, 325 (2013)

    PubMed  PubMed Central  Article  Google Scholar 

  12. Walhout, A. J. M. What does biologically meaningful mean? A perspective on gene regulatory network validation. Genome Biol. 12, 109 (2011)

    PubMed  PubMed Central  Article  Google Scholar 

  13. Kim, W.-C., Kim, J.-Y., Ko, J.-H., Kang, H. & Han, K.-H. Identification of direct targets of transcription factor MYB46 provides insights into the transcriptional regulation of secondary wall biosynthesis. Plant Mol. Biol. 85, 589–599 (2014)

    CAS  PubMed  Article  Google Scholar 

  14. Ahnert, S. E. Power graph compression reveals dominant relationships in genetic transcription networks. Mol. Biosyst. 9, 2681–2685 (2013)

    CAS  PubMed  Article  Google Scholar 

  15. Kim, W.-C. et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis. Plant J. 73, 26–36 (2013)

    CAS  PubMed  Article  Google Scholar 

  16. del Pozo, J. C., Diaz-Trivino, S., Cisneros, N. & Gutierrez, C. The balance between cell division and endoreplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis. Plant Cell 18, 2224–2235 (2006)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. del Pozo, J. C., Boniotti, M. B. & Gutierrez, C. Arabidopsis E2Fc functions in cell division and is degraded by the ubiquitin-SCFAtSKP2 pathway in response to light. Plant Cell 14, 3057–3071 (2002)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. de Jager, S. M., Menges, M., Bauer, U. M. & Murray, J. A. H. Arabidopsis E2F1 binds a sequence present in the promoter of S-phase-regulated gene AtCDC6 and is a member of a multigene family with differential activities. Plant Mol. Biol. 47, 555–568 (2001)

    CAS  PubMed  Article  Google Scholar 

  19. Mariconti, L. et al. The E2F family of transcription factors from Arabidopsis thaliana: novel and conserved components of the retinoblastoma/E2F pathway in plants. J. Biol. Chem. 277, 9911–9919 (2002)

    CAS  PubMed  Article  Google Scholar 

  20. Kosugi, S. & Ohashi, Y. Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Physiol. 128, 833–843 (2002)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. de Jager, S. et al. Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc. Plant Mol. Biol. 71, 345–365 (2009)

    CAS  PubMed  Article  Google Scholar 

  22. Heckmann, S. et al. The E2F transcription factor family regulates CENH3 expression in Arabidopsis thaliana. Plant J. 68, 646–656 (2011)

    CAS  PubMed  Article  Google Scholar 

  23. del Pozo, J. C., Diaz-Trivino, S., Cisneros, N. & Gutierrez, C. The E2FC-DPB transcription factor controls cell division, endoreplication and lateral root formation in a SCFSKP2A-dependent manner. Plant Signal. Behav. 2, 273–274 (2007)

    PubMed  PubMed Central  Article  Google Scholar 

  24. Yamaguchi, M. et al. VND-INTERACTING2, a NAC domain transcription factor, negatively regulates xylem vessel formation in Arabidopsis. Plant Cell 22, 1249–1263 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Yamaguchi, M. et al. VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. Plant Physiol. 153, 906–914 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Wenkel, S., Emery, J., Hou, B.-H., Evans, M. M. S. & Barton, M. K. A feedback regulatory module formed by LITTLE ZIPPER and HD-ZIPIII genes. Plant Cell 19, 3379–3390 (2007)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Zhong, R. & Ye, Z.-H. MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol. 53, 368–380 (2012)

    CAS  PubMed  Article  Google Scholar 

  28. Ohashi-Ito, K., Oda, Y. & Fukuda, H. Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that govern programmed cell death and secondary wall formation during xylem differentiation. Plant Cell 22, 3461–3473 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Pyo, H., Demura, T. & Fukuda, H. TERE; a novel cis-element responsible for a coordinated expression of genes related to programmed cell death and secondary wall formation during differentiation of tracheary elements. Plant J. 51, 955–965 (2007)

    CAS  PubMed  Article  Google Scholar 

  30. Dinneny, J. R. et al. Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942–945 (2008)

    ADS  CAS  PubMed  Article  Google Scholar 

  31. Iyer-Pascuzzi, A. S. et al. Cell identity regulators link development and stress responses in the Arabidopsis root. Dev. Cell 21, 770–782 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M. & Birnbaum, K. D. Cell-specific nitrogen responses mediate developmental plasticity. Proc. Natl Acad. Sci. USA 105, 803–808 (2008)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. Brady, S. M. et al. A stele-enriched gene regulatory network in the Arabidopsis root. Mol. Syst. Biol. 7, 459 (2011)

    PubMed  PubMed Central  Article  Google Scholar 

  34. Gong, W. et al. Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol. 135, 773–782 (2004)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Paz-Ares, J. & The REGIA Consortium. REGIA, an EU project on functional genomics of transcription factors from Arabidopsis thaliana. Comp. Funct. Genomics 3, 102–108 (2002)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Underwood, B. A., Vanderhaeghen, R., Whitford, R., Town, C. D. & Hilson, P. Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations. Plant Biotechnol. J. 4, 317–324 (2006)

    CAS  PubMed  Article  Google Scholar 

  37. Yamada, K., Lim, J., Dale, J. & Chen, H. Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302, 842–846 (2003)

    ADS  CAS  PubMed  Article  Google Scholar 

  38. Pruneda-Paz, J. L., Breton, G., Para, A. & Kay, S. A. A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science 323, 1481–1485 (2009)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Kubo, M. et al. Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19, 1855–1860 (2005)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Hawker, N. P. & Bowman, J. L. Roles for class III HD-Zip and KANADI genes in Arabidopsis root development. Plant Physiol. 135, 2261–2270 (2004)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Updegraff, D. Semimicro determination of cellulose in biological materials. Anal. Biochem. 32, 420–424 (1969)

    CAS  PubMed  Article  Google Scholar 

  42. Scott, T. A. & Melvin, E. H. Determination of dextran with anthrone. Anal. Chem. 25, 1656–1661 (1953)

    CAS  Article  Google Scholar 

  43. Liu, Y., Burch-Smith, T., Schiff, M., Feng, S. & Dinesh-Kumar, S. P. Molecular chaperone Hsp90 associates with resistance protein N and its signaling proteins SGT1 and Rar1 to modulate an innate immune response in plants. J. Biol. Chem. 279, 2101–2108 (2004)

    CAS  PubMed  Article  Google Scholar 

  44. Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003)

    CAS  PubMed  Article  Google Scholar 

  45. Walley, J. W. et al. Mechanical stress induces biotic and abiotic stress responses via a novel cis-element. PLoS Genet. 3, e172 (2007)

    PubMed Central  Article  CAS  Google Scholar 

  46. Nusinow, D. A. et al. The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth. Nature 475, 398–402 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Pearson, R. D. et al. puma: a Bioconductor package for propagating uncertainty in microarray analysis. BMC Bioinformatics 10, 211 (2009)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. Mordelet, F. & Vert, J.-P. SIRENE: supervised inference of regulatory networks. Bioinformatics 24, i76–i82 (2008)

    PubMed  Article  Google Scholar 

  49. Huynh-Thu, V. A., Irrthum, A., Wehenkel, L. & Geurts, P. Inferring regulatory networks from expression data using tree-based methods. PLoS ONE 5, e12776 (2010)

    ADS  PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. Greenfield, A., Madar, A., Ostrer, H. & Bonneau, R. DREAM4: Combining genetic and dynamic information to identify biological networks and dynamical models. PLoS ONE 5, e13397 (2010)

    ADS  PubMed  PubMed Central  Article  CAS  Google Scholar 

  51. Haury, A.-C., Mordelet, F., Vera-Licona, P. & Vert, J.-P. TIGRESS: Trustful Inference of Gene REgulation using Stability Selection. BMC Syst. Biol. 6, 145 (2012)

    PubMed  PubMed Central  Article  Google Scholar 

  52. Küffner, R., Petri, T., Tavakkolkhah, P., Windhager, L. & Zimmer, R. Inferring gene regulatory networks by ANOVA. Bioinformatics 28, 1376–1382 (2012)

    PubMed  Article  CAS  Google Scholar 

  53. Marbach, D. et al. Wisdom of crowds for robust gene network inference. Nature Methods 9, 796–804 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We thank M. Tierney (University of Vermont) for 35S::GFP seeds, T. Demura for VND7 resources, M.K. Barton for REV:GR seeds, E.P. Spalding for advice on manuscript revision, and C. Gutierrez for E2Fc RNAi and E2Fc N-terminal deletion overexpressor seeds and useful discussion. This research was supported by the Office of Science (BER) Department of Energy Grant DE-FG02-08ER64700DE (to S.P.H. and S.A.K.), National Institute of General Medical Sciences of the National Institutes of Health under award numbers RO1GM056006 and RC2GM092412 (to S.A.K.), National Institute of Health (R01GM107311) and National Science Foundation (IOS-1036491 and IOS-1352478) to K.D., USDA CRIS 1907-21000-030 to D.W. and L.F., a Royal Society UK Fellowship (to S.E.A.), and UC Davis Startup Funds and a Hellman Fellowship (to S.M.B).

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Authors and Affiliations

Authors

Contributions

M.T.-T., L.L. and M.d.L. contributed equally to this work. T.W.T. and A.G. contributed equally to this work. S.M.B. and S.P.H. contributed equally to this work. M.T.-T., L.L., M.d.L., S.M.B., and S.P.H. designed the research. M.T.-T., L.L., M.d.L., A.G., G.X., N.F.Y., G.M.T., M.T.V., R.L., P.P.H., C.W., and K.D. performed the research. M.T.-T., L.L., G.T., T.W.T., N.T., J.C., M.P., D.K., I.T., S.E.A., S.M.B. and S.P.H. analysed the data. L.Z., D.W., G.B., J.L.P.-P., and S.A.K. contributed new reagents/analytic tools. M.T.-T., L.L., G.M.T., S.M.B. and S.P.H. wrote the article. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to S. P. Hazen or S. M. Brady.

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Extended data figures and tables

Extended Data Figure 1 Number of novel and previously described protein–DNA interactions and transcription factors involved in secondary cell wall biosynthesis and xylem development.

a, b, Venn diagrams of overlap between previously reported19 interactions (a) or transcription factors (b) and those of the xylem-specific gene regulatory network. *Includes genes that were not included in the yeast one hybrid screen.

Extended Data Figure 2 Activation or repression of VND7 by E2Fc is dynamic and dose-dependent.

a, Intensity of LUC bioluminescence quantified using Andor Solis image analysis software. Data are means ± s.d. (n = 20). Asterisks denote significance at P < 0.05 determined by Student’s t-test. b, Quantitative PCR with reverse transcription of E2Fc and VND7 transcripts in ΔN-E2Fc (E2Fc overexpressor line lacking the N-terminal domain) expressing plants versus Col-0 control. Red dashed line marks the point at which VND7 is unchanged compared to control. Each data point is an individual biological replicate with 3 technical replicates. c, 3-week-old tobacco leaves were infiltrated with the p19 silencing inhibitor and either the reporter VND7p::GUS or VND7p::GUS and either 35S::E2Fc::MYC or 35S::RBR::GFP, or both. Extracted protein was then used in a quantitative MUG fluorescent assay, where relative fluorescence was measured 60 min after incubation with substrate. Data are means ± s.d., n = 3.

Extended Data Figure 3 Binding of NST2 and SND1 to fragments of CESA7, CESA8, and KOR promoters.

af, Electrophoretic mobility shift assays showing NST2 (ad) and SND1 (ef) protein specifically binds the promoters of cellulose-associated genes. Probe was incubated in the absence or presence of GST or GST:SND1 protein extracts. The arrowheads indicate the specific protein–DNA complexes, while arrows indicate free probe.

Extended Data Figure 4 Sub-networks of network genes differentially expressed in response to iron deprivation of high salinity.

a, b, Sub-network of genes with q values of ≤ 0.01 and whose fold change between mean expression values was ≥ 1.5 in either direction in iron deprivation (a) or high NaCl (b) stress microarray data set. Nodes are coloured according to in-degree as shown on scale bars below sub-networks. Transcription factors with the highest in-degree are labelled and indicated with a black circle.

Extended Data Figure 5 The reconstructed gene regulatory consensus network based on analysis of the iron-deprivation expression data set by different network inference methods.

a, Unsupervised; b, supervised in the first pass; c, supervised after the validated two connections have been added in the training set. Edge transparency denotes P ≤ 0.06 for the Pearson correlation coefficient (PCC); edge width is proportional to PCC; edge value corresponds to the total edge score; a greater value corresponds to a more significant score. Yellow and red nodes correspond to transcription factor and target gene nodes, respectively; black and blue edges denote Y1H-derived and inferred interactions, respectively.

Extended Data Figure 6 Iron deprivation and NaCl stress influences lignin and phenylpropanoid biosynthesis associated gene expression.

a, No change was observed in the expression of 4CL1::GFP in 4 days after imbibition (DAI) roots transferred to a control media (left, n = 4) or media with 140 mM NaCl for 48 h (right, n = 4). b, Increased fuchsin staining of xylem cells as well as of cell walls of non-vascular cells in 4 DAI roots transferred to a control media (left) or media with an iron chelator for 72 h (right). c, No change was observed in the expression of VND7::YFP in 4 DAI roots transferred to a control media (left, n = 4) or media with an iron chelator for 72 h (right, n = 5).

Extended Data Figure 7 The reconstructed gene regulatory consensus network based on analysis of the salt-stress expression data set by different network inference methods.

a, Unsupervised; b, supervised in the first pass; c, supervised after the validated two connections have been added in the training set. Edge transparency denotes P ≤ 0.06 for the Pearson correlation coefficient (PCC); edge width is proportional to PCC; edge value corresponds to the total edge score; a greater value corresponds to a more significant score. Yellow and red nodes correspond to transcription factor and target gene nodes, respectively; black and blue edges denote Y1H-derived and inferred interactions, respectively.

Extended Data Figure 8 Schematic diagram of dual-luciferase reporter vector development.

a, Three distinct donor vectors harbouring either the transcription factor, VP64 activation domain fused to the 35S minimal promoter, or a promoter fragment. b, The dual reporter vector, pLAH-LARm, is then recombined with the three donor vectors to generate the single reporter vector (c).

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Taylor-Teeples, M., Lin, L., de Lucas, M. et al. An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517, 571–575 (2015). https://doi.org/10.1038/nature14099

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